EP0533719B1 - Verfahren für den nachweis und/oder die bestimmung von hormonen - Google Patents

Verfahren für den nachweis und/oder die bestimmung von hormonen Download PDF

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EP0533719B1
EP0533719B1 EP91910511A EP91910511A EP0533719B1 EP 0533719 B1 EP0533719 B1 EP 0533719B1 EP 91910511 A EP91910511 A EP 91910511A EP 91910511 A EP91910511 A EP 91910511A EP 0533719 B1 EP0533719 B1 EP 0533719B1
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Prior art keywords
antibody
medium
antibodies
assay
polyclonal
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EP0533719A1 (de
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Marie-Christine Maurel
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Centre National de la Recherche Scientifique CNRS
Institut National de la Recherche Agronomique INRA
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Centre National de la Recherche Scientifique CNRS
Institut National de la Recherche Agronomique INRA
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/963—Methods of stopping an enzyme reaction or stabilizing the test materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to a method for the detection and / or determination of hormones including placental hormones.
  • hormones are meant the substances produced by the various human and animal endocrine glands and in particular, pituitary hormones (FSH, LH, TSH, GH, ACTH, prolactin, oxytocin, MSH, ADH), related placental hormones [human choriogonadotropins ( hCG) and equine (eCG), choriosomatomammotropin (CS), placental GH], thyroid hormones, adrenal hormones, gonadal hormones and pancreatic hormones.
  • FSH pituitary hormones
  • LH pituitary hormones
  • TSH GH
  • ACTH prolactin
  • oxytocin MSH
  • ADH related placental hormones
  • hCG human choriogonadotropins
  • eCG equine
  • CS choriosomatomammotropin
  • placental GH thyroid hormones
  • adrenal hormones gonadal hormones and pancreatic hormones.
  • hormones derived from amino acids (thyroid hormones, hormones of the adrenal medulla), peptide and protein hormones (pancreatic hormones, GH, ACTH, prolactin, MSH, ADH, oxytocin ), steroid hormones (gonadal hormones, adrenal cortex hormones) and glycoprotein hormones (LH, FSH, TSH, hCG, eCG).
  • Glycoprotein hormones are characterized by the same structural organization: they are formed by the association of two subunits ⁇ and ⁇ .
  • the ⁇ subunit is identical for all glycoprotein hormones of the same animal species. Conversely, the ⁇ subunit is different from one hormone to another and confers specificity of action.
  • Peptide hormones similarly, have a structural relationship as well as polypeptide hormones (example: GH and prolactin).
  • these hormones are not secreted at constant rates but, on the contrary, exhibit variations in circulating concentration of low amplitude (micropulsatility) or, in certain cases, high amplitude (ex: peak secretion of FSH then LH in preovulatory period). It is therefore necessary to have assay methods which allow precise and specific measurement of the circulating level of these different hormones, in the blood or in any other possible biological fluid.
  • the detection and / or determination of the gonadotropic hormones LH and FSH is particularly important in the study of the physiology of male and female reproduction in animals and humans.
  • the functioning of the ovary during the period of genital activity involves a succession of ovarian cycles during which the female gametes mature at regular intervals.
  • the ovarian cycle has two phases: the follicular phase (growth of the follicle and maturation of the oocyte) leading to ovulation, a period conducive to fertilization, and the luteal phase (formation of the corpus luteum).
  • FSH is responsible for the growth and maturation of the preovulatory follicle; LH intervenes more particularly in the terminal follicular growth of the latter and in the triggering of ovulation.
  • ovulation occurs at a very precise and constant time, for each species, after the LH peak (from 17 to 24 hours depending on the species).
  • CG choriogonadotropin
  • the assay methods currently used in human clinics for these different hormones are, on the one hand, radioimmunological methods (for example: “AMERLEX-M” system from AMERSHAM, “MAIAclone” system from SERONO for hLH, hFSH, hTSH, hCG, etc.) and on the other hand, enzymoimmunometric or immunometric methods (using a non-enzymatic marker: latex particles, europium chelate).
  • radioimmunological methods for example: "AMERLEX-M” system from AMERSHAM, “MAIAclone” system from SERONO for hLH, hFSH, hTSH, hCG, etc.
  • enzymoimmunometric or immunometric methods using a non-enzymatic marker: latex particles, europium chelate.
  • enzyme-immunometric systems some use a fluorescent signal (enzyme-microparticulate system “IMX” from ABBOTT; “STRATUS SYSTEM” from AMERICAN DADE for LH, FSH, hCG ”) or luminescent (system “AMERLITE” from AMERSHAM for LH, TSH, FSH, hCG; “MAGIC LITE” system from CIBA CORNING for TSH, prolactin ).
  • IMX enzyme-microparticulate system "IMX” from ABBOTT; "STRATUS SYSTEM” from AMERICAN DADE for LH, FSH, hCG
  • luminescent system “AMERLITE” from AMERSHAM for LH, TSH, FSH, hCG
  • MAGIC LITE CIBA CORNING for TSH, prolactin
  • the first antibody (AC 1 ) binding to the LH of the sample or to the radioactive LH * added (incubation from 36 to 48 h).
  • the separation of the antibody-LH complex from the free LH * is carried out using the second antibody (AC 2 ) immobilized on the walls of a tube (incubation: 3 hours, with shaking at 37 ° C).
  • the AC1-sample incubation is 16 to 24 h at 20 ° C with shaking; the addition of LH-peroxidase requires a further incubation for 16 to 24 h at 4 ° C.
  • the revelation is carried out using TMB (30 min to 2 h).
  • the detection threshold obtained with the short protocol is 260 pg / ml and that obtained with the long protocol is 70 pg / ml.
  • the methods for assaying FSH in animals are essentially radioimmunological, as are the other hormones.
  • the subject of the present invention is a method for the detection and / or immunological determination of human or animal pituitary hormones, in culture media or in biological fluids, using at least two antibodies, specific for the hormone to be assayed , which method is characterized in that in order to increase the sensitivity and the specificity of said detection and / or of said assay, said antibodies are, prior to their implementation in said detection or said assay, preincubated in a medium containing plasma or serum devoid of the hormone to be detected and / or to be measured, obtained by hypophysectomy (medium INC).
  • preincubated antibody is meant, within the meaning of the present invention, an "exhausted” antibody, - by contacting or passage through a suitable chromatography column -, by any substance which can induce non-hormonal and non-specific serum interference.
  • the biological fluid is advantageously serum, plasma, whole blood, milk or urine.
  • the medium containing serum or plasma, devoid of the hormone to be detected and / or to be assayed, is called INC medium.
  • said INC medium is associated with a buffer at neutral pH and with a surfactant.
  • the INC medium comprises a serum or a plasma devoid of pituitary hormones, obtained by hypophysectomy of an animal and a buffer at neutral pH associated with a surfactant.
  • a buffer at neutral pH associated with a surfactant and in particular a PBS-Tween-BSA buffer.
  • the INC medium comprises a serum or a plasma of hypophysectomized ram and a buffer at neutral pH, associated with a surfactant.
  • the plasma or serum devoid of the hormone to be assayed and the buffer are in a 1: 1 ratio.
  • said method is advantageously an enzymo-immunometric test, highly specific and sensitive, for detecting at least one hormone.
  • the first antibody, fixed on a solid support, saturated in INC medium and the second antibody, optionally coupled to an appropriate enzyme, preincubated in INC medium are brought into contact with the biological fluid to be tested, after which the enzymatic activity linked to the solid and / or free phase is revealed by any suitable means.
  • the revelation of the reaction is then carried out by the introduction of a third antibody coupled to a appropriate enzyme, preincubated in said INC medium and binding specifically to the second antibody.
  • the first and second antibodies are advantageously chosen from the group which comprises anti-FSH, anti-LH, anti-TSH, anti-GH, anti-ACTH, anti-prolactin, anti-oxytocin, anti monoclonal antibodies -ADH, anti-MSH and anti-FSH, anti-LH, anti-TSH, anti-GH, anti-ACTH, anti-prolactin, anti-oxytocin, anti-ADH and anti-MSH polyclonal antibodies.
  • the anti-LH polyclonal antibodies are in particular obtained by immunization of an appropriate animal using an antigen comprising an equimolar mixture of different purified fractions of ovine LH listed 1051, 1055, 1072, 1063, 1085 and 1086, then by purification of the immuniserum obtained by ion exchange chromatography or by affinity chromatography, which polyclonal antibodies have a percentage cross-reactions with other glycoprotein hormones such as FSH, less than 4%, that is to say specificity with respect to LH, in that they exhibit a polyspecificity for recognition of LH numerous animal species and in particular at least ovine, bovine, caprine, porcine, canine, camelina, murine as well as deer species and in that they exhibit an affinity for ovine LH of the order of 10 9 M -1 , respectively in two different animal species.
  • FSH glycoprotein hormones
  • the first and second antibodies are advantageously chosen from the group which comprises rabbit anti-LH anti-polyclonal antibodies and polyclonal horse anti-ovine LH antibodies and the third antibody is advantageously chosen from the group which comprises anti-Horse IgG and anti-Rabbit IgG coupled to an appropriate enzyme.
  • the first antibody is a rabbit polyclonal antibody to sheep LH
  • the second antibody is a horse polyclonal antibody to sheep LH
  • the third antibody is a horse IgG coupled to an appropriate enzyme and in particular to a peroxidase or to a ⁇ -galactosidase.
  • the various ovine LH isoforms are preferably in equimolar amounts.
  • a suitable purification of said anti-LH polyclonal antibodies can be carried out, as the case may be, either by affinity chromatography or by ion exchange chromatography.
  • the immunological assay and more particularly the enzymo-immunometric assay of the hormones has both the characteristics of a quantitative assay by providing very good sensitivity as well as very good reproducibility, and d '' a qualitative mix by its ease of realization and interpretation.
  • this assay has both the characteristics of a quantitative assay by providing very good sensitivity (detection threshold: 60 pg / ml) as well as very good reproducibility, and of a qualitative assay by its ease of realization and interpretation. It can therefore be used both in the laboratory for very precise dosages (e.g. measurement of the micropulsatility of the secretion of LH) than in the field (detection of the preovulatory LH peak for the artificial insemination of synchronized females and / or superovulated females, embryo donors).
  • This LH assay usable in many species: bovine, ovine, caprine, porcine, camelina, canine, murine, cervidae, allows the precise detection of the preovulatory LH peak in the female and presents an obvious physiological and agronomic interest. It will also allow a good estimate of the optimal time chosen for artificial insemination in each species concerned. In fact, the interval between the LH peak and ovulation is constant for each species; on the other hand, the time interval separating the arrival in heat (benchmark used until now) and the peak of LH is very fluctuating according to the individuals of the same race.
  • the test in accordance with the invention therefore proves to be an essential tool for properly controlling this factor of variability which is important for the successful fertilization of oocytes and the collection of good quality embryos. Indeed, knowing the precise moment of ovulation, thanks to the detection of the LH peak, and of artificial insemination, we can deduce at what stage of development will be the embryos that we want to collect. This point is important insofar as embryos that are too young or too advanced cannot be marketed (eg with a broken film membrane).
  • this animal LH assay test is interesting in the context of the development of a new treatment (synchronization, superovulation) or the extension of these treatments to a breed, or even to a species whose precovulatory physiology is poorly known (e.g. cervids).
  • the enzymo-immunometric assay of human LH has both the characteristics of a quantitative dosage by the very good sensitivity acquired by said process and a qualitative dosage by its ease of implementation and interpretation also acquired by the process according to the invention. It can therefore be used in the laboratory or at home for individual use ("Home test"); in the latter case, it will serve as a kit for detecting the preovulatory LH peak, characterized by the appearance of an unambiguous green color, and making it possible to predict the time of ovulation in women.
  • the antibodies when it is desired to carry out an assay having a species specificity, the antibodies must have particular qualities; in this case, the choice of antibodies is important for an appropriate implementation of the method according to the invention. It is for this purpose that the Applicant has also developed antibodies which are particularly suitable for the process according to the invention.
  • the present invention also relates to a reagent for the implementation of the method of detection and / or immunological assay of hormones in animals including humans, according to the invention, characterized in that it comprises an anti-hormone antibody suitable for being used as second and / or third antibody in said method, which antibody is preincubated in an INC medium.
  • the present invention also relates to a reagent for the implementation of the method of detection and / or immunological assay of hormones in animals including humans, according to the invention, characterized in that it comprises an anti-hormone antibody suitable for being used as the first antibody in said method, which first antibody is fixed on a solid support, saturated in INC medium.
  • the enzyme is chosen from the group which comprises peroxidases and ⁇ -galactosidase.
  • the peroxidase revealing substrates are advantageously OPD and ABTS; the substrate for revealing ⁇ -galactosidase is advantageously 4-methylumbelliferyl- ⁇ -D-galactopyranoside (MUG) and ortho-nitrophenyl- ⁇ -O-galactopyranoside (ONPG).
  • MUG 4-methylumbelliferyl- ⁇ -D-galactopyranoside
  • ONPG ortho-nitrophenyl- ⁇ -O-galactopyranoside
  • the solid support is advantageously chosen from known supports such as microtitration plates, sticks, strips, beads or membranes.
  • the solid phase used is, without limitation, a 96-well microtiter plate in Luxlon (hydrophobic plastic), but the assay can be carried out on other types of plates or other types of supports, in particular sticks, strips, beads or membranes.
  • EXAMPLE 5 Determination of animal LHs with the AP2-peroxidase system .
  • An LH assay can be carried out under the same conditions as those of Example 4, in which AP2 is coupled to a peroxidase.
  • the tests carried out gave a dose-response curve of less good sensitivity than that obtained according to the protocol described above but sufficient for the detection of high concentrations of LH (from 15 ng / ml).
  • the incubation of the substrate must in return be longer 30 to 45 min if necessary for the detection of preovulatory peaks.
  • FIG. 17 shows the LH secretion profile in ng / ml as a function of time in a doe (doe 80). The results obtained in all of the females again show that the moment of the LH peak is very variable during the heat season: it generally takes place at the start of the heat.
  • the assays are carried out as described in Example 3, from daily blood samples taken from 2 bitches during the heat season.
  • the preovulatory LH peak extends over a longer period of time than in the other described species: the duration is 24 hours on average. Therefore, it can be detected by only taking a daily blood sample, which is an advantage for the customers of a veterinary practice.
  • the results obtained with the "DIXIE" dog illustrated in FIG. 18 clearly show this. The tests are read with the naked eye as for the other species.
  • FIG. 18 represents the secretion profile of LH, (- ⁇ -) and of progesterone (- * -) in ng / ml, obtained in the dog DIXIE.
  • the kit Given the length of the heat period, in the dog, the kit will allow to know precisely the moment of ovulation in the dog followed and will allow to practice insemination at the optimal time (fertilization takes place 48 hours after the ovulation, in the dog). Again, an insemination scheme can be established precisely, using the method according to the invention.
  • FIG. 19 illustrates a profile of LH secretion in ng / ml obtained with an assay according to the invention (- * -) and with an RIA assay (- ⁇ -).

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Claims (19)

  1. Verfahren zur Detektion und/oder quantitativen immunologischen Bestimmung von Hormonen der Hypophyse von Menschen oder Tieren in Kulturmedien oder in biologischen Flüssigkeiten, bei dem mindestens zwei Antikörper eingesetzt werden, die für das quantitativ zu bestimmende Hormon spezifisch sind, dadurch gekennzeichnet, daß zur Erhöhung der Sensibilität und der Spezifität der Detektion und/oder der quantitativen Bestimmung die Antikörper vor ihrem Einsatz für die Detektion oder die quantitative Bestimmung in einem Medium vorinkubiert werden, welches Plasma oder Serum, das frei ist von dem zu detektierenden und/oder quantitativ zu bestimmenden Hormon und durch Hypophysektomie erhalten worden ist (INC-Medium), enthält.
  2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß das INC-Medium mit einem pH-neutralen Puffer und einem grenzflächenaktiven Mittel assoziiert wird.
  3. Verfahren nach Anspruch 1 oder Anspruch 2, dadurch gekennzeichnet, daß das INC-Medium ein von Hypophysehormonen freies und durch Hypophysektomie eines Tieres erhaltenes Serum oder Plasma und einen pH-neutralen Puffer, assoziiert mit einem grenzflächenaktiven Mittel, enthält.
  4. Verfahren nach Anspruch 3, dadurch gekennzeichnet , daß das INC-Medium ein Serum oder ein Plasma eines hypophysektomisierten Widders und einen pH-neutralen Puffer, assoziiert mit einem grenzflächenaktiven Mittel, enthält.
  5. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß das Plasma oder Serum, das frei ist von dem quantitativ zu bestimmenden Hormon, und der Puffer in einem Verhältnis von 1:1 vorliegen.
  6. Verfahren nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß der erste Antikörper, fixiert auf einem festen Träger, gesättigt in INC-Medium, und der zweite Antikörper, gegebenenfalls gekuppelt an ein geeignetes Enzym, vorinkubiert in einem INC-Medium, mit der zu untersuchenden biologischen Flüssigkeit in Kontakt gebracht werden und darauf die an die feste Phase gebundene und/oder freie enzymatische Aktivität mit jedem geeigneten Mittel nachgewiesen wird.
  7. Verfahren nach Anspruch 6, dadurch gekennzeichnet, daß, wenn der zweite in dem INC-Medium vorinkubierte Antikörper nicht an ein Enzym gekuppelt ist, der Nachweis der Reaktion durch Einführung eines dritten Antikörpers erfolgt, der an ein geeignetes Enzym gekuppelt und in dem INC-Medium vorinkubiert ist und der sich spezifisch an den zweiten Antikörper bindet.
  8. Verfahren nach Anspruch 6 oder Anspruch 7, dadurch gekennzeichnet, daß der erste und der zweite Antikörper vorteilhafterweise aus der Gruppe ausgewählt werden, die die monoclonalen Anti-FSH-, Anti-LH-, Anti-TSH-, Anti-GH-, Anti-ACTH-, Anti-Prolactin-, Anti-Ocytocin-, Anti-ADH-, Anti-MSH-Antikörper und die polyclonalen Anti-FSH-, Anti-LH-, Anti-TSH-, Anti-GH-, Anti-ACTH-, Anti-Prolactin-, Anti-Ocytocin-, Anti-ADH- und Anti-MSH-Antikörper umfaßt.
  9. Verfahren nach Anspruch 8, dadurch gekennzeichnet, daß die polyclonalen Anti-LH-Antikörper vor allem erhalten werden durch Immunisierung eines geeigneten Tieres mit Hilfe eines Antigens, das ein äquimolares Gemisch verschiedener gereinigter Fraktionen von Schaf-LH, registriert unter der Nr. 1051, 1055, 1072, 1063, 1085 und 1086, umfaßt und anschließend durch Reinigung des durch Ionenaustauschchromatographie oder durch Affinitätschromatographie erhaltenen Immunserums, wobei die polyclonalen Antikörper einen Prozentsatz an Kreuzreaktionen mit den anderen Glykoprotein-Hormonen, wie FSH, von weniger als 4%, d.h. eine Spezifität gegenüber LH aufweisen, daß sie eine Polyspezifität der Erkennung von LH von zahlreichen Tierarten, insbesondere mindestens der Arten Schafe, Rinder, Ziegen, Schweine, Hunde, Maus sowie Hirsche aufweisen und eine Affinität gegenüber Schaf-LH in der Größenordnung von 109M-1 jeweils in zwei unterschiedlichen Tierarten aufweisen.
  10. Verfahren nach einem der Ansprüche 6 bis 9, dadurch gekennzeichnet, daß der erste und der zweite Antikörper vorteilhafterweise aus der Gruppe ausgewählt werden, die polyclonale Kaninchen-Anti-LH-Schaf-Antikörper und polyclonale Pferd-Anti-LH-Schaf-Antikörper umfaßt, und daß der dritte Antikörper vorteilhafterweise aus der Gruppe ausgewählt wird, die die Pferd-Anti-IgG und die Kaninchen-Anti-IgG, gekuppelt an ein geeignetes Enzym, umfaßt.
  11. Verfahren nach einem der Ansprüche 6 bis 10, dadurch gekennzeichnet, daß der erste Antikörper ein polyclonaler Kaninchen-Anti-LH-Schaf-Antikörper ist, der zweite Antikörper ein polyclonaler Pferd-Anti-LH-Schaf-Antikörper ist und der dritte Antikörper ein Pferd-Anti-IgG, gekuppelt an ein geeignetes Enzym und insbesondere an eine Peroxidase oder an eine β-Galactosidase, ist.
  12. Reagens zur Durchführung des Verfahrens der Detektion und/oder der quantitativen immunologischen Bestimmung von Hormonen beim Tier einschließlich des Menschen nach einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, daß es einen Anti-Hormon-Antikörper enthält, der als zweiter und/oder dritter Antikörper in dem genannten Verfahren verwendet werden kann, und daß der Antikörper in einem INC-Medium vorinkubiert wird.
  13. Reagens für die Durchführung des Verfahrens der Detektion und/oder der quantitativen immunologischen Bestimmung von Hormonen beim Tier einschließlich des Menschen nach einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, daß es einen Anti-Hormon-Antikörper enthält, der als erster Antikörper in dem Verfahren verwendet werden kann, wobei der erste Antikörper auf einem festen Träger, gesättigt in INC-Medium, fixiert ist.
  14. Besteck für die Detektion und/oder Diagnose von Hypophysehormonen (FSH, LH, TSH, GH, ACTH, Prolactin, Ocytocin, ADH, MSH) bei Menschen oder beim Tier oder Kit für die Durchführung des Tests gemäß einem der Ansprüche 1 bis 11, dadurch gekennzeichnet, daß es zusätzlich zu den nützlichen Mengen Puffer für die Ausführung der Detektion umfaßt:
    - einen ersten für das quantitativ zu bestimmende Hormon spezifischen Antikörper, ausgewählt aus den Anti-quantitativ zu bestimmendes Hormon-Antikörpern, fixiert auf einem festen Träger, gesättigt in INC-Medium, nach Anspruch 13;
    - einen zweiten für das quantitativ zu bestimmende Hormon spezifischen Antikörper, ausgewählt aus den Antispezifisch zu bestimmendes Hormon-Antikörpern, gekuppelt an ein geeignetes Enzym und vorinkubiert in dem INC-Medium gemäß Anspruch 12;
    - ein Substrat für den Nachweis des Enzyms und
    - ein INC-Medium.
  15. Besteck nach Anspruch 14, dadurch gekennzeichnet, daß es, wenn für die quantitative Bestimmung von LH eingesetzt, umfaßt:
    - einen ersten polyclonalen Antikörper, fixiert auf einem festen Träger, gesättigt in INC-Medium, ausgewählt unter den polyclonalen Kaninchen-Anti-LH-Schaf-Antikörpern und den polyclonalen Pferd-Anti-LH-Schaf-Antikörpern;
    - ein Enzym-Antikörperkonjugat, in dem der Antikörper ausgewählt ist aus der Gruppe, die die polyclonalen Kaninchen-Anti-LH-Schaf-Antikörper und die polyclonalen Pferd-Anti-LH-Schaf-Antikörper umfaßt, wobei die Konjugate in einem INC-Medium vorinkubiert worden sind;
    - ein Substrat für den Nachweis des Enzyms;
    - eine Entnahmekapillare; und
    - ein INC-Medium.
  16. Besteck nach Anspruch 15, dadurch gekennzeichnet, daß das Enzym aus der Gruppe, umfassend die Peroxidasen und β-Galactosidase, ausgewählt ist.
  17. Besteck nach Anspruch 14, dadurch gekennzeichnet, daß es umfaßt:
    - eine Reihe von geeigneten festen Trägern, überzogen mit einem ersten Antikörper, ausgewählt unter den Antiquantitativ zu bestimmendes Hormon-Antikörpern, wobei die festen Träger in INC-Medium gesättigt sind;
    - einen zweiten Antikörper, ausgewählt aus der Gruppe, die die Antikörper Anti-FSH, Anti-LH, Anti-TSH, Anti-GH, Anti-ACTH, Anti-Prolactin, Anti-Ocytocin und Anti-ADH, die von dem ersten Antikörper verschieden sind, vorinkubiert in einem INC-Medium, enthält;
    - ein Peroxidase-Anti-IgG-Konjugat, vorinkubiert in einem INC-Medium;
    - ein Substrat für den Nachweis der Peroxidase; und
    - ein INC-Medium.
  18. Besteck nach Anspruch 17, dadurch gekennzeichnet, daß es umfaßt:
    - eine Reihe von festen Trägern, überzogen mit einem ersten Antikörper, ausgewählt aus der Gruppe, umfassend die polyclonalen Kaninchen-Anti-LH-Schaf-Antikörper, wobei die festen Träger in INC-Medium gesättigt sind;
    - einen zweiten Antikörper, ausgewählt aus der Gruppe, umfassend die polyclonalen Pferd-Anti-LH-Antikörper;
    - ein Peroxidase-Pferd-Anti-IgG-Konjugat, vorinkubiert in einem INC-Medium;
    - ein Substrat für den Nachweis der Peroxidase;
    - eine Entnahmekapillare; und
    - ein INC-Medium.
  19. Besteck nach Anspruch 17, dadurch gekennzeichnet, daß es umfaßt:
    - eine Reihe von festen Trägern, überzogen mit einem ersten Antikörper, ausgewählt aus der Gruppe, umfassend die polyclonalen Antikörper von Kaninchen-Anti-LH-Schaf, wobei die festen Träger in INC-Medium gesättigt sind;
    - ein Konjugat β-Galactosidase-polyclonale Pferd-Anti-LH-Schaf-Antikörper, vorinkubiert in einem INC-Medium;
    - ein Substrat für den Nachweis der β-Galactosidase;
    - eine Entnahmekapillare; und
    - ein INC-Medium.
EP91910511A 1990-06-01 1991-05-30 Verfahren für den nachweis und/oder die bestimmung von hormonen Expired - Lifetime EP0533719B1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9006863 1990-06-01
FR9006863A FR2662804B1 (fr) 1990-06-01 1990-06-01 Anticorps polyclonaux anti-lh et leurs applications a la detection et/ou au dosage de la lh, chez de multiples especes animales.
PCT/FR1991/000427 WO1991019195A2 (fr) 1990-06-01 1991-05-30 Methode de detection et/ou de dosage des hormones et anticorps utilisables dans ladite methode de detection

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EP0533719B1 true EP0533719B1 (de) 1997-01-29

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IE (1) IE911894A1 (de)
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PT (1) PT97839B (de)
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JP3479100B2 (ja) * 1993-06-02 2003-12-15 帝国臓器製薬株式会社 免疫化学的簡易半定量方法および装置
WO1998048273A1 (en) * 1997-04-23 1998-10-29 Harrison Richard J Method for fertility detection
US20010023070A1 (en) * 1998-05-29 2001-09-20 Reinhard Ebner Interleukins-21 and 22
EP1143250A3 (de) * 2000-04-03 2004-11-17 Inverness Medical Switzerland GmbH Testverfahren und Vorrichtungen für Analytisoformen
JP2001349892A (ja) * 2000-04-03 2001-12-21 Unilever Nv 検査方法及びデバイス
EP1143249B1 (de) * 2000-04-03 2014-01-15 Alere Switzerland GmbH Testverfahren und Vorrichtungen für Analytisoformen
MXPA02010643A (es) * 2000-04-26 2004-05-17 Land O Lakes Inc Un metodo de tratamiento de proteinas de soya y un producto de proteina de soya producido mediante este metodo.
WO2006075966A1 (en) * 2005-01-17 2006-07-20 Gyros Patent Ab A versatile flow path
US8592219B2 (en) * 2005-01-17 2013-11-26 Gyros Patent Ab Protecting agent
WO2006076371A2 (en) * 2005-01-10 2006-07-20 Baylor College Of Medicine Method of quantitative immunohistochemistry and in situ hybridization
WO2010005738A1 (en) 2008-06-16 2010-01-14 Duke University Chemical sensors and methods for making and using the same
US9958442B2 (en) * 2009-02-11 2018-05-01 Duke University Sensors incorporating antibodies and methods of making and using the same
CN102297966A (zh) * 2011-05-27 2011-12-28 天津农学院 一种半定量检测孕酮胶体金试纸条及其制备方法
WO2013153291A1 (fr) 2012-04-12 2013-10-17 Repropharm Procede et trousse de detection du pic preovulatoire de lh
US9688754B2 (en) 2014-02-20 2017-06-27 Alder Biopharmaceuticals, Inc. Anti-ACTH antibodies and use thereof
WO2015187227A2 (en) 2014-03-13 2015-12-10 Duke University Electronic platform for sensing and control of electrochemical reactions
CN105445256A (zh) * 2014-09-02 2016-03-30 江苏泽成生物技术有限公司 促黄体生成素lh定量测定试剂盒及其制备方法与检测方法
CN105424937B (zh) * 2015-10-05 2017-11-21 华中农业大学 一种猴早期妊娠检测的孕酮胶体金免疫层析试纸条
JP6959883B2 (ja) * 2018-02-28 2021-11-05 株式会社あすか製薬メディカル オキシトシンの定量方法

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DK0533719T3 (da) 1997-08-11
WO1991019195A2 (fr) 1991-12-12
PT97839B (pt) 1998-10-30
AU7958091A (en) 1991-12-31
NZ238394A (en) 1993-04-28
EP0533719A1 (de) 1993-03-31
PT97839A (pt) 1992-02-28
WO1991019195A3 (fr) 1992-01-09
FR2662804B1 (fr) 1994-05-27
ES2099746T3 (es) 1997-06-01
US5674700A (en) 1997-10-07
ATE148562T1 (de) 1997-02-15
FR2662804A1 (fr) 1991-12-06
ZA914121B (en) 1992-03-25
DE69124511T2 (de) 1997-07-31
IE911894A1 (en) 1991-12-04
DE69124511D1 (de) 1997-03-13

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